Prophylactic activity of orally administered dry-heat-sterilized Acremonium egyptiacum against Trypanosoma congolense-induced animal African trypanosomosis.

Animal African trypanosomosis (AAT) is an important global disease of livestock that causes economic losses of up to 4.5 billion US dollars per year. Thus, eliminating AAT in endemic countries will improve agricultural productivity and economic growth. To prevent AAT, vector control and the development of prophylactic drugs are crucial. Ascofuranone (AF) is a bioactive fungal compound with proven in vitro trypanocidal potency and in vivo treatment efficacy. However, the complex stereoselective synthesis of AF has prevented its cost-effective industrial production. Recently, a genetically modified strain of Acremonium egyptiacum fungus that produces a high yield of AF was developed. Therefore, we hypothesized that the oral administration of the AF-producing fungus itself may be effective against AAT. Hence, this study aimed to evaluate the prophylactic activity of orally administered dry-heat-sterilized A. egyptiacum against Trypanosoma congolense IL3000 infection using a mouse model. The survival rate was significantly prolonged (p = 0.009), and parasitemia was suppressed in all AF-fungus-treated groups (Group 1-9) compared with that in the untreated control group (Group 10). Hence, the trypanocidal activity of AF was retained after dry-heat-sterilization of the AF-producing fungus and that its oral administration effectively prevented AAT. Since AAT is endemic to rural areas with underdeveloped veterinary infrastructure, dry-heat-sterilized A. egyptiacum would be the most cost-effective potential treatment for AAT.

Mitigation of Trypanosoma congolense-Associated Anemia and Expression of Trans-sialidase (TconTS) Gene Variants by Eugenol.

PURPOSE: African Animal Trypanosomosis (AAT) caused by Trypanosoma congolense is a parasitic disease affecting the livestock industry in sub-Saharan Africa and usually results in severe anemia, organ damage, and ultimately the death of the infected host. The present study was designed to investigate the possible chemotherapeutic effect of eugenol on T. congolense infections and its inhibitory effect on the trans-sialidase (TconTS) gene expression. METHODS: Animals were infected with T. congolense and treated with 15 and 30 mg/kg body weight (BW) of eugenol for ten (10) days. RESULTS: The eugenol (15 mg/kg BW) significantly (P < 0.05) reduced the T. congolense proliferation, increased animal survival, and reduced serum urea level. However, both dosages of eugenol significantly (P < 0.05) ameliorated T. congolense-induced anemia, renal hypertrophy, splenomegaly, and reduced total damage score in the liver and kidney of infected animals. In addition, the compound significantly (P < 0.05) downregulated the expression levels of TconTS1, TconTS2, TconTS3, and TconTS4 but the effect was more pronounced (sevenfold reduction) on TconTS1. CONCLUSIONS: The oral administration of eugenol suppressed T. congolense proliferation and prevented some major pathologies associated with trypanosomiasis infection. The reversal of renal hypertrophy and splenomegaly by the compound in addition to the reduction in the expression level of the TconTS gene variants could explain the observed anemia ameliorative potential of the compound.

Phloroglucinol as a Potential Candidate against Trypanosoma congolense Infection: Insights from In Vivo, In Vitro, Molecular Docking and Molecular Dynamic Simulation Analyses.

Sub-Saharan Africa is profoundly challenged with African Animal Trypanosomiasis and the available trypanocides are faced with drawbacks, necessitating the search for novel agents. Herein, the chemotherapeutic potential of phloroglucinol on T. congolense infection and its inhibitory effects on the partially purified T. congolense sialidase and phospholipase A2 (PLA2) were investigated. Treatment with phloroglucinol for 14 days significantly (p < 0.05) suppressed T. congolense proliferation, increased animal survival and ameliorated anemia induced by the parasite. Using biochemical and histopathological analyses, phloroglucinol was found to prevent renal damages and splenomegaly, besides its protection against T. congolense-associated increase in free serum sialic acids in infected animals. Moreover, the compound inhibited bloodstream T. congolense sialidase via mixed inhibition pattern with inhibition binding constant (Ki) of 0.181 µM, but a very low uncompetitive inhibitory effects against PLA2 (Ki > 9000 µM) was recorded. Molecular docking studies revealed binding energies of -4.9 and -5.3 kcal/mol between phloroglucinol with modeled sialidase and PLA2 respectively, while a 50 ns molecular dynamics simulation using GROMACS revealed the sialidase-phloroglucinol complex to be more compact and stable with higher free binding energy (-67.84 ± 0.50 kJ/mol) than PLA2-phloroglucinol complex (-77.17 ± 0.52 kJ/mol), based on MM-PBSA analysis. The sialidase-phloroglucinol complex had a single hydrogen bond interaction with Ser453 while none was observed for the PLA2-phloroglucinol complex. In conclusion, phloroglucinol showed moderate trypanostatic activity with great potential in ameliorating some of the parasite-induced pathologies and its anti-anemic effects might be linked to inhibition of sialidase rather than PLA2.

Antitrypanosomal activity of hydromethanol extract of leaves of Cymbopogon citratus and seeds of Lepidium sativum: in-vivo mice model.

BACKGROUND: Trypanosomiasis is one of the neglected tropical diseases of both humans and animals which decreases their productivity and causes death in the worst scenario. Unavailability of vaccines, the low therapeutic index of trypanocidal drugs, and the development of resistance lead to the need for research focused on developing alternative treatment options especially from medicinal plants. The present study was aimed to investigate antitrypanosomal activities of leaves of Cymbopogon citratus and seeds of Lepidium sativum in in-vivo mice model. METHODS: The plant extracts were prepared by maceration using 80% methanol and reconstituted with 10% dimethyl sulfoxide (DMSO) to have the desired concentration. The test doses were adjusted to 100, 200 and 400 mg/kg based on the toxicity profile. The plants extracts were administered to the respective groups of mice after the 12th day of field isolate T. congolense inoculation for seven consecutive days. The level of parasitemia, bodyweight, packed cell volume (PCV), and differential white blood cell counts were measured. RESULTS: The in -vivo test results revealed that both plant extracts had dose-dependent antitrypanosomal activity. Both crude extracts showed a significant reduction in parasite load (P < 0.05), increased or prevent the fall of PCV value (P < 0.05), decreased lymphocytosis and increased neutrophil counts (p < 0.05) and improved bodyweight but significant bodyweight increment (P < 0.05) was observed only in C. citratus treated mice compared to the negative and positive controls. CONCLUSION: The present study concluded that the crude extracts of leaves of C. citratus and seeds of L. sativum had antitrypanosomal effects. Both plants extracts reduced parasitemia level, prevented anemia and improved bodyweight of treated mice. Comparative results from all tested parameters showed that the best activities were observed with C. citratus treated groups of mice.

Divergent metabolism between Trypanosoma congolense and Trypanosoma brucei results in differential sensitivity to metabolic inhibition.

Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon sensitivity to metabolic inhibition. Like the bloodstream stage of T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly lower in bloodstream stage T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Transcriptomics analysis showed higher levels of transcripts associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation, and the glycosomal succinate shunt in T. congolense, compared to T. brucei. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and saturated fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with metabolic inhibitors, confirming that electron transport chain activity is not essential in T. congolense. However, the parasite exhibits increased sensitivity to inhibition of mitochondrial pyruvate import, compared to T. brucei. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold higher EC50 for the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions.

Diminazene resistance in Trypanosoma congolense is not caused by reduced transport capacity but associated with reduced mitochondrial membrane potential.

Trypanosoma congolense is a principal agent causing livestock trypanosomiasis in Africa, costing developing economies billions of dollars and undermining food security. Only the diamidine diminazene and the phenanthridine isometamidium are regularly used, and resistance is widespread but poorly understood. We induced stable diminazene resistance in T. congolense strain IL3000 in vitro. There was no cross-resistance with the phenanthridine drugs, melaminophenyl arsenicals, oxaborole trypanocides, or with diamidine trypanocides, except the close analogs DB829 and DB75. Fluorescence microscopy showed that accumulation of DB75 was inhibited by folate. Uptake of [3 H]-diminazene was slow with low affinity and partly but reciprocally inhibited by folate and by competing diamidines. Expression of T. congolense folate transporters in diminazene-resistant Trypanosoma brucei brucei significantly sensitized the cells to diminazene and DB829, but not to oxaborole AN7973. However, [3 H]-diminazene transport studies, whole-genome sequencing, and RNA-seq found no major changes in diminazene uptake, folate transporter sequence, or expression. Instead, all resistant clones displayed a moderate reduction in the mitochondrial membrane potential Ψm. We conclude that diminazene uptake in T. congolense proceed via multiple low affinity mechanisms including folate transporters; while resistance is associated with a reduction in Ψm it is unclear whether this is the primary cause of the resistance.

Trypanosuppressive effects of Kolaviron may be associated with down regulation of Trypanothione reductase in Trypanosoma congolense infection.

Trypanothione reductase is a key enzyme that upholds the redox balance in hemoflagellate protozoan parasites such as T. congolense. This study aims at unraveling the potency of Kolaviron against trypanothione reductase in T. congolense infection using Chrysin as standard. The experiment was performed using three different approaches; in silico, in vitro and in vivo. Kolaviron and Chrysin were docked against trypanothione reductase, revealing binding energies (-9.3 and -9.0 kcal/mol) and Ki of 0.211µM and 0.151µM at the active site of trypanothione reductase as evident from the observed strong hydrophobic/hydrogen bond interactions. Parasitized blood was used for parasite isolation and trypanothione reductase activity assay using standard protocol. Real-time PCR (qPCR) assay was implored to monitor expression of trypanothione reductase using primers targeting the 177-bp repeat satellite DNA in T. congolense with SYBR Green to monitor product accumulation. Kolaviron showed IC50 values of 2.64µg/ml with % inhibition of 66.78 compared with Chrysin with IC50 values of 1.86µg/ml and % inhibition of 53.80. In vivo studies following the administration of these compounds orally after 7 days post inoculation resulted in % inhibition of Chrysin (57.67) and Kolaviron (46.90). Equally, Kolaviron relative to Chrysin down regulated the expression trypanothione reductase gene by 1.352 as compared to 3.530 of the infected group, in clear agreement with the earlier inhibition observed at the fine type level. Overall, the findings may have unraveled the Kolaviron potency against Trypanosoma congolense infection in rats.

Veterinary trypanocidal benzoxaboroles are peptidase-activated prodrugs.

Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism.

The therapeutic potential of phytol towards Trypanosoma congolense infection and the inhibitory effects against trypanosomal sialidase.

The search for novel therapeutic candidates against animal trypanosomiasis is an ongoing scientific endevour because of the negative impacts of the disease to the African livestock industry. In this study, the in vivo therapeutic potentials of phytol toward Trypanosoma congolense infection and the inhibitory effects on trypanosomal sialidase were investigated. Rats were infected with T. congolense and administered daily oral treatment of 50 and 100 mg/kg BW of phytol. Within the first 10 days of the treatment, no antitrypanosomal activity was recorded but a moderate trypanostatic activity was observed from day 17-day 21 pi. However, at 100 mg/kg BW, phytol demonstrated a significant (p < 0.05) ameliorative potentials toward T. congolense-induced host-associated pathological damages such as anaemia, hepatic and renal damages; and the data was comparable to diminazine aceturate. Moreover, the T. congolense caused a significant (p < 0.05) increase in free serum sialic acid level which was significantly (p < 0.05) prevented in the presence of phytol (100 mg/kg BW). In an in vitro analysis, phytol inhibited partially purified T. congolense sialidase using an uncompetitive inhibition pattern with inhibition binding constant of 261.24 µmol/mL. Subsequently, molecular docking revealed that the compound binds to homology modelled trypanosomal sialidase with a binding free energy of -6.7 kcal/mol which was mediated via a single hydrogen bond while Trp324 and Pro274 were the critical binding residues. We concluded that phytol has moderate trypanostatic activity but with a great potential in mitigating the host-associated cellular damages while the anaemia amelioration was mediated, in part, through the inhibition of sialidase.

Molecular identification of diminazene aceturate-resistant strains of Trypanosoma congolense in naturally infected domestic animals of Yoko in the centre region of Cameroon.

African animal trypanosomiases (AAT) remain the major constraint for livestock production, agriculture and food security in Africa. Although several control measures have been developed to fight AAT, the use of trypanocides remains the main strategy in most affected poor and rural communities. However, several studies have highlighted drug-resistant-trypanosome infections in many African countries, though this phenomenon is still not well described. This study aims to detect trypanosome species and the molecular profiles of drug-resistant-trypanosomes in naturally infected domestic animals of Yoko in the centre region of southern Cameroon. Therefore, in October 2017, 348 animals were blood sampled. The level of packed cell volume (PCV) was evaluated in each animal and trypanosome infections were investigated with the capillary tube centrifugation technique (CTC). Thereafter, DNA was extracted from blood samples and different trypanosome species were identified by PCR. The resistant/sensitive molecular profiles of trypanosomes for diminazene aceturate (DA) and isometamidium chloride (ISM) were investigated by PCR-RFLP. About 18.4% (64/348) of animals analyzed by PCR were found with trypanosome infections including Trypanosoma vivax, Trypanosoma brucei s.l. and Trypanosoma congolense forest and savannah. Trypanosoma congolense savannah was the predominant species with an infection rate of 15.2%. Between villages, significant (p˂0.0001) differences were found in the overall trypanosome infection rates. No molecular profile for ISM resistant-trypanosomes was identified. Conversely, about 88.9% (40/45) of T. congolense positive samples have shown molecular profiles of DA-resistant strains while the remaining 11.1% (5/45) showed mixed molecular profiles of resistant/sensitive strains. Results showed that the molecular profiles of DA-resistant strains of T. congolense in domestic animals of Yoko were widespread. This data needs to be confirmed by testing in vivo the drug susceptibilities of the trypanosome strains herein detected. In conclusion, appropriate future control measures are required. In addition to the intensification of vector control, ISM is advised for the treatment of animals infected by trypanosomes.

Hyaluronidase inhibitory saponins and a trypanocidal isoflavonoid from the aerial parts of Oxytropis lanata.

A chemical examination of an extract from the aerial part of Oxytropis lanata led to the isolation and identification of 36 compounds, including saponins, isoflavonoids, oxazoles, and glycosides. The three among them were previously unreported oleanane-type saponins. In trypanocidal screening, 5,7,4'-trihydroxyisoflavone showed inhibitory activity against Trypanosoma congolense (IC50 = 10.5 µM), the causative agent of African trypanosomosis in animals; this activity was similar to that of active compounds from the roots of this plant. O. lanata is known to be a traditional medicinal plant in Mongolia for the treatment of inflammatory diseases. The anti-hyaluronidase effect of saponins 3, 5, 8, and 9, (IC50 = 0.15-0.22 mM) was stronger than that of sodium cromoglicate, which was used as a reference drug (IC50 = 0.37 mM). The chemical structures of the new saponins were determined based on HRFABMS, 1H and 13C NMR, 1H-1H COSY, HMQC, HMBC, and ROESY spectroscopic data along with chemical procedures.

Evaluation of the relative roles of the Tabanidae and Glossinidae in the transmission of trypanosomosis in drug resistance hotspots in Mozambique.

BACKGROUND: Tsetse flies (Diptera: Glossinidae) and tabanids (Diptera: Tabanidae) are haematophagous insects of medical and veterinary importance due to their respective role in the biological and mechanical transmission of trypanosomes. Few studies on the distribution and relative abundance of both families have been conducted in Mozambique since the country's independence. Despite Nicoadala, Mozambique, being a multiple trypanocidal drug resistance hotspot no information regarding the distribution, seasonality or infection rates of fly-vectors are available. This is, however, crucial to understanding the epidemiology of trypanosomosis and to refine vector management. METHODS: For 365 days, 55 traps (20 NGU traps, 20 horizontal traps and 15 Epsilon traps) were deployed in three grazing areas of Nicoadala District: Namitangurine (25 traps); Zalala (15 traps); and Botao (15 traps). Flies were collected weekly and preserved in 70% ethanol. Identification using morphological keys was followed by molecular confirmation using cytochrome c oxidase subunit 1 gene. Trap efficiency, species distribution and seasonal abundance were also assessed. To determine trypanosome infection rates, DNA was extracted from the captured flies, and submitted to 18S PCR-RFLP screening for the detection of Trypanosoma. RESULTS: In total, 4379 tabanids (of 10 species) and 24 tsetse flies (of 3 species), were caught. NGU traps were more effective in capturing both the Tabanidae and Glossinidae. Higher abundance and species diversity were observed in Namitangurine followed by Zalala and Botao. Tabanid abundance was approximately double during the rainy season compared to the dry season. Trypanosoma congolense and T. theileri were detected in the flies with overall infection rates of 75% for tsetse flies and 13% for tabanids. Atylotus agrestis had the highest infection rate of the tabanid species. The only pathogenic trypanosome detected was T. congolense. CONCLUSIONS: Despite the low numbers of tsetse flies captured, it can be assumed that they are still the cyclical vectors of trypanosomosis in the area. However, the high numbers of tabanids captured, associated to their demonstrated capacity of transmitting trypanosomes mechanically, suggest an important role in the epidemiology of trypanosomosis in the Nicoadala district. These results on the composition of tsetse and tabanid populations as well as the observed infection rates, should be considered when defining strategies to control the disease.

In vitro and in vivo antitrypanosomal efficacy of combination therapy of Anogeissus leiocarpus, Khaya senegalensis and potash.

ETHNOPHARMACOLOGICAL RELEVANCE: Pastoralists in Nigeria mix barks of Anogeissus leiocarpus (AL) Khaya senegalensis (KS) and potash (Pt) to treat animal African trypanosomosis. AIM: To evaluate antitrypanosomal potential of A. leiocarpus, K. senegalensis and potash for insights into the traditional claim of antitrypanosomal combination therapy (ATCT). MATERIALS AND METHODS: Fifty microliter each of six different concentrations of AL, KS, Pt, AL + KS, AL + KS + Pt and diminazene aceturate (DA, positive control) was incubated with 50 µL of parasite-laden blood containing 108Trypanosoma congolense cells in a 96-well microtitre plate. Negative control wells were devoid of the extracts and drug but supplemented with phosphate-buffered saline (PBS). Efficacy of treatment was observed at 1 h interval for complete immobilisation or reduced motility of the parasites. Each incubated mixture was inoculated into mouse at the point of complete immobilisation of parasite motility or at the end of 6-h observation period for concentrations that did not immobilise the parasites completely. For in vivo assessment, thirty-five parasitaemic rats were randomly allocated into seven groups of 5 rats each. Each rat in groups I-V was treated with 500 mg/kg of AL, KS, Pt, AL + KS and AL + KS + Pt, respectively, for 7 days. Rats in groups VI and VII were treated with diminazene aceturate 3.5 mg/kg once and PBS 2 mL/kg (7 days), which served as positive and negative controls, respectively. Daily monitoring of parasitaemia through the tail vein, packed cell volume and malondialdehyde were used to assess efficacy of the treatments. RESULTS: The AL + KS + Pt group significantly (p < 0.05) and dose-dependently reduced parasite motility and completely immobilized the parasites at 10, 5 and 2.5 µg/µL with an IC50 of 9.1×10-4 µg/µL. All the mice with conditions that produced complete cessation of parasite motility did not develop parasitaemia within one month of observation. The AL + KS group significantly (p < 0.05) lowered the level of parasitaemia and MDA, and significantly (p < 0.05) maintained higher PCV than PBS group. CONCLUSION: The combination of A. leiocarpus and K. senegalensis showed better antitrypanosomal effects than single drug treatment and offers prospects for ATCT. Our findings support ethnopharmacological use of combined barks of A. leiocarpus and K. senegalensis by pastoralist in the treatment of animal African trypanosomosis in Nigeria.

Effect of vegetable oils on the experimental infection of mice with Trypanosoma congolense.

Vegetable oils are frequently used as solvents for lipophilic materials; accordingly, the effects of their components should be considered in animal experiments. In this study, the effects of various vegetable oils on the course of Trypanosoma congolense infection were examined in mice. C57BL/6J mice were orally administered four kinds of oils (i.e., coconut oil, olive oil, high oleic safflower oil, and high linoleic safflower oil) with different fatty acid compositions and infected with T. congolense IL-3000. Oil-treated mice infected with T. congolense showed significantly higher survival rates and lower parasitemia than those of control mice. Notably, coconut oil, which mainly consists of saturated fatty acids, delayed the development of parasitemia at the early stage of infection. These results indicated that vegetable oil intake could affect T. congolense infection in mice. These findings have important practical implications; for example, they suggest the potential effectiveness of vegetable oils as a part of the regular animal diet for controlling tropical diseases and indicate that vegetable oils are not suitable solvents for studies of the efficacy of lipophilic agents against T. congolense.

European propolis is highly active against trypanosomatids including Crithidia fasciculata.

Extracts of 35 samples of European propolis were tested against wild type and resistant strains of the protozoal pathogens Trypanosoma brucei, Trypanosoma congolense and Leishmania mexicana. The extracts were also tested against Crithidia fasciculata a close relative of Crithidia mellificae, a parasite of bees. Crithidia, Trypanosoma and Leishmania are all members of the order Kinetoplastida. High levels of activity were obtained for all the samples with the levels of activity varying across the sample set. The highest levels of activity were found against L. mexicana. The propolis samples were profiled by using liquid chromatography with high resolution mass spectrometry (LC-MS) and principal components analysis (PCA) of the data obtained indicated there was a wide variation in the composition of the propolis samples. Orthogonal partial least squares (OPLS) associated a butyrate ester of pinobanksin with high activity against T. brucei whereas in the case of T. congolense high activity was associated with methyl ethers of chrysin and pinobanksin. In the case of C. fasciculata highest activity was associated with methyl ethers of galangin and pinobanksin. OPLS modelling of the activities against L. mexicana using the mass spectrometry produced a less successful model suggesting a wider range of active components.

Trypanostatic activity of geranylacetone: Mitigation of Trypanosoma congolense-associated pathological pertubations and insight into the mechanism of anaemia amelioration using in vitro and in silico models.

Trypanosoma congolense is an important pathogen that wreaks havoc in the livestock industry of the African continent. This study evaluated the in vivo antitrypanosomal activity of geranylacetone and its ameliorative effect on the disease-induced anaemia and organ damages as well as its inhibitory effects against trypanosomal sialidase using in vitro and in silico techniques. Geranylacetone was used to treat T. congolense infected rats, at a dose of 50 and 100 mg/kg BW, for 14 days where it was found to reduce the parasite burden in the infected animals. Moreover, 100 mg/kg BW of geranylacetone significantly (p < 0.05) ameliorated the anaemia, hepatic and renal damages caused by the parasite. This is in addition to the alleviation of the parasite-induced hepatosplenomegaly and upsurge in free serum sialic acid levels in the infected animals which were associated with the observed anaemia amelioration by the compound. Consequently, bloodstream T. congolense sialidase was partially purified on DEAE cellulose column and inhibition kinetic studies revealed that the enzyme was inhibited by geranylacetone via an uncompetitive inhibition pattern. In silico analysis using molecular docking with Autodock Vina indicated that geranylacetone binds to trypanosomal sialidase with a minimum free binding energy of -5.8 kcal/mol which was mediated by 26 different kinds of non-covalent interactions excluding hydrogen bond whilst Asp163 and Phe421 had the highest number of the interactions. The data suggests that geranylacetone has trypanostatic activity and could protect animals against the T. congolense-induced anaemia through the inhibition of sialidase and/or the protection of the parasite-induced hepatosplenomegaly.

Acylated Lignans Isolated from Brachanthemum gobicum and Their Trypanocidal Activity.

Eight isovaleryllignans (1-4 and 8-11), three isovalerylphenylpropanoids (5-7), three known lignans (12-14), and four known compounds were isolated from an extract of the aerial part of Brachanthemum gobicum. The structures of the isolated compounds were elucidated based on NMR and MS data analyses. The enantiomers of compounds 1-3, 5, 8, and 9 were isolated using chiral-phase HPLC, and the absolute configurations of 1a/1b-3a/3b, 5a/5b, 8a/8b, and 9a/9b were elucidated from their optical rotations and ECD spectra; the other lignans were assumed to be racemic or scalemic by chiral-phase HPLC analyses and optical rotation data. Some of the acylated lignans (racemic mixtures) (1-4, 8, 9, and 12-14) exhibited moderate inhibitory activities against Trypanosoma congolense, the causative agent of nagana disease in animals.

Parasite specific 7SL-derived small RNA is an effective target for diagnosis of active trypanosomiasis infection.

Human and animal African trypanosomiasis (HAT & AAT, respectively) remain a significant health and economic issue across much of sub-Saharan Africa. Effective control of AAT and potential eradication of HAT requires affordable, sensitive and specific diagnostic tests that can be used in the field. Small RNAs in the blood or serum are attractive disease biomarkers due to their stability, accessibility and available technologies for detection. Using RNAseq, we have identified a trypanosome specific small RNA to be present at high levels in the serum of infected cattle. The small RNA is derived from the non-coding 7SL RNA of the peptide signal recognition particle and is detected in the serum of infected cattle at significantly higher levels than in the parasite, suggesting active processing and secretion. We show effective detection of the small RNA in the serum of infected cattle using a custom RT-qPCR assay. Strikingly, the RNA can be detected before microscopy detection of parasitaemia in the blood, and it can also be detected during remission periods of infection when no parasitaemia is detectable by microscopy. However, RNA levels drop following treatment with trypanocides, demonstrating accurate prediction of active infection. While the small RNA sequence is conserved between different species of trypanosome, nucleotide differences within the sequence allow generation of highly specific assays that can distinguish between infections with Trypanosoma brucei, Trypanosoma congolense and Trypanosoma vivax. Finally, we demonstrate effective detection of the small RNA directly from serum, without the need for pre-processing, with a single step RT-qPCR assay. Our findings identify a species-specific trypanosome small RNA that can be detected at high levels in the serum of cattle with active parasite infections. This provides the basis for the development of a cheap, non-invasive and highly effective diagnostic test for trypanosomiasis.

Novel Minor Groove Binders Cure Animal African Trypanosomiasis in an in Vivo Mouse Model.

Animal African trypanosomiasis (AAT) is a significant socioeconomic burden for sub-Saharan Africa because of its huge impact on livestock health. Existing therapies including those based on minor groove binders (MGBs), such as the diamidines, which have been used for decades, have now lost efficacy in some places because of the emergence of resistant parasites. Consequently, the need for new chemotherapies is urgent. Here, we describe a structurally distinct class of MGBs, Strathclyde MGBs (S-MGBs), which display excellent in vitro activities against the principal causative organisms of AAT: Trypanosoma congolense, and Trypanosoma vivax. We also show the cure of T. congolense-infected mice by a number of these compounds. In particular, we identify S-MGB-234, compound 7, as curative by using two applications of 50 mg/kg intraperitoneally. Crucially, we demonstrate that S-MGBs do not show cross-resistance with the current diamidine drugs and are not internalized via the transporters used by diamidines. This study demonstrates that S-MGBs have significant potential as novel therapeutic agents for AAT.

The individual components of commercial isometamidium do not possess stronger trypanocidal activity than the mixture, nor bypass isometamidium resistance.

The four components present in the trypanocidal treatment Samorin, the commercially available formulation of isometamidium, were separated and purified by column chromatography. These compounds as well as the Samorin mixture and the other phenanthridine trypanocide, homidium, were tested on Trypanosoma congolense and wild type, diamidine- and isometamidium-resistant Trypanosoma brucei brucei strains using an Alamar blue drug sensitivity assay. EC50 values obtained suggest that M&B4180A (2) was the most active of the components, followed by M&B38897 (1) in all the strains tested, whereas M&B4596 (4) was inactive. Samorin was found to be significantly more active than any of the individual components alone, against T. congolense and all three T. b, brucei strains. Samorin and all its active constituents displayed reduced activity against the previously characterised isometamidium-resistant strain ISMR1.